The invention relates to peptides that have a stable backbone that can be readily adapted to provide a multitude of interactive domains such as inhibitory or binding domains.
One drawback to immunologically based diagnostic assays is the reliance on the use of antibodies. These reagents, whether monoclonal or polyclonal, are large macromolecular polypeptides that are expensive to produce and often become unstable during storage, necessitating a short shelf life for many diagnostic products. In addition, a typical immunoglobin (e.g.-IgG) contains a great deal of mass (the Fc region) that is physiologically important, but that plays no role in antigen recognition. Such added mass is unnecessary for many applications and can add background noise, inhibit diffusion and cause side reactions. Moreover, the disulfide bonds holding heavy and light chains of antibodies together are potentially labile. Thus, only a small fraction of the antibody structure (and therefore mass) is directly involved in antigen recognition, yet the entirety of the antibody is often produced and used in a sensor or diagnostic device.
It is possible to produce smaller Fab regions from intact antibodies, but Fab production requires several chemical or enzymatic processing steps and additional protein purification procedures. Such processing procedures add significant costs to the diagnostic product.
What is needed is an easily synthesized, stable antigen recognition element, where a higher proportion of the molecular mass is involved in antigen recognition.
The invention provides easily synthesized, peptide backbones that can be readily modified to include binding domains, inhibitor domains, linkers, labels, reagents, reactive sites, catalytic sites or reagents and other chemical entities.
In one embodiment, the invention provides a stable isolated peptide comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NO:2-6, 8-11 or 14. Such a stable isolated peptide can have a polyproline helix, a short loop region, and an alpha helix, where the peptide folds so that the polyproline helix and the alpha helix hydrophobically interact. Peptides of the invention are often more stable than a peptide having SEQ ID NO:1, which is a small peptide derived from Avian Pancreatic Polypeptide. Other peptides of the invention are less stable than a peptide having SEQ ID NO:1. Desirable peptides include an amino acid sequence with at least 90% identity to SEQ ID NO:11 or 14. Peptides having SEQ ID NO:11 or 14, are folded as described above and further stabilized by a disulfide bond.
The invention also provides isolated nucleic acids encoding a stable peptide comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NO:2-6, 8-11 or 14. Preferably, the isolated nucleic acid encodes and amino acid sequence with at least 90% identity to any one of SEQ ID NO:11 or 14. Examples of such nucleic acids comprise SEQ ID NO:12 or 13.
In another embodiment, the invention provides a peptide-based reagent comprising a peptide backbone and an interactive domain, where the peptide backbone comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NO:2-6, 8-11 or 14. Desirable peptide-based reagents have a peptide backbone and an interactive domain, where the peptide backbone comprises an amino acid sequence with at least 90% identity to SEQ ID NO:11 or 14. The peptide backbone can have a polyproline helix, a short loop region, and an alpha helix, where the peptide backbone folds so that the polyproline helix and the alpha helix hydrophobically interact. Desirable peptide backbones are more stable than a peptide having SEQ ID NO:1. Desirable peptide-based reagents are more stable than the peptide backbone that does not have the interactive domain. However, insertion of some interactive domains de-stabilizes the peptide backbone and such destabilized peptide-based reagents may still be useful to one of skill in the art.
The interactive domains for attachment onto, or insertion into, the peptide backbone can be any useful peptide or molecule chosen by one of skill in the art. Examples of interactive domains include binding domains, inhibitor domains, antigen-recognizing peptides, linkers, labels, solid supports, and enzymatic active sites. One peptide-based reagent of the invention has an interactive domain where the peptide comprises SEQ ID NO:18.
The invention also provides a method comprising:
defining a search zone comprising a site of interaction on a target protein to which a peptide can interact;
defining a size for the peptide;
defining a class of amino acids for each position in the amino acid sequence of the peptide;
substituting each member of a defined class of amino acids into each position of the amino acid sequence of the peptide sequence to generate an output library file comprising a plurality of output peptide sequences;
communicating the output library file to a molecular docking program to fit each of the plurality of output peptide sequences to the search zone and to create a target protein-peptide sequence fit score;
ranking the plurality of output peptides sequences by target protein-peptide sequence fit score; and
displaying each of the plurality of output peptide sequences and its associated target protein-peptide sequence fit score;
wherein a portion of the plurality of output peptide sequences can stably interact with the target protein.
The search zone can include the x-, y-, and z-coordinates of each non-hydrogen atoms in the target protein. Output peptide sequences with higher target protein-peptide sequence fit scores can often bind with higher affinity to the target protein. The method can further include receiving an input percentage selection to limit the plurality of output peptide sequences to a certain percentage; wherein the input percentage selection is capable of limiting an output library file size and a library complexity. Each class of amino acids can separately comprise any one of genetically encoded L-amino acids, naturally occurring non-genetically encoded L-amino acids, synthetic L-amino acids, D-enantiomers of genetically encoded amino acids, D-enantiomers of naturally occurring non-genetically encoded amino acids, or synthetic D-amino acids. Each class of amino acids can also separately comprise any one of hydrophilic amino acids, hydrophobic amino acids, cysteine-like amino acids, acidic amino acids, basic amino acids, polar amino acids, aromatic amino acids, apolar amino acids or aliphatic amino acids. In one embodiment, the target protein is bovine pancreatic trypsin and one of the output peptide sequences is YKLKY (SEQ ID NO:18).
The invention is also directed to a system for creating peptide sequences, comprising: a processor; a memory coupled to the processor; a display couple to the processor; a make peptide sequence component capable of executing on the processor to generate peptide sequences; an output class component capable of executing on the processor to display each class of amino acid residues used by the make peptide sequence component; and an output peptide sequence component capable of executing on the processor to display peptide sequences. One example of a display is a printer. The output class component may be capable of displaying each class of amino acid residues used by the make peptide sequence component.
The invention further provides a machine-accessible medium having associated content capable of directing the machine to perform a method, the method comprising:
receiving a search zone comprising a plurality of coordinates for atoms in an target site to which a plurality of peptides can bind with varying affinities;
receiving a peptide length parameter comprising a number of amino acids;
receiving a defined class of amino acid structures to be analyzed for fitness at each position along the peptide length;
generating a output library file comprising a plurality of output peptide sequences containing each amino acid from each defined class of amino acid structures at each position along the peptide length;
sequentially translating and rotating each member of the class of amino acid structures at each position within a peptide relative to the search zone to sequentially create a peptide sequence with a target site-peptide sequence fit score;
ranking peptide sequences by target site-peptide sequence fit scores; and
displaying a selected percentage of the target site-peptide sequence fit scores with the associated peptide sequences.
The method performed by the machine-accessible medium can further include displaying labels for the output peptide sequences and storing the search zone.